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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 呂紹俊(Shao-Chun Lu) | |
| dc.contributor.author | Yi-Ting Hsieh | en |
| dc.contributor.author | 謝依庭 | zh_TW |
| dc.date.accessioned | 2021-06-14T16:43:13Z | - |
| dc.date.available | 2009-09-11 | |
| dc.date.copyright | 2008-09-11 | |
| dc.date.issued | 2008 | |
| dc.date.submitted | 2008-08-01 | |
| dc.identifier.citation | 1. Lauritzen, L., et al., The essentiality of long chain n-3 fatty acids in relation to development and function of the brain and retina. Prog Lipid Res, 2001. 40(1-2): p. 1-94.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/40242 | - |
| dc.description.abstract | 1-acyl-sn-glycerol-3-phosphate acyltransferase (AGPAT)又稱為 lysophosphatidic acid acyltransferase (LPAAT)，是催化脂肪酸由acyl-CoA (通常為不飽和脂肪酸) 鍵結到1-acyl-sn-glycerol-3-phosphate的sn-2上產生phosphatidic acid (PA)的酵素。因此AGPAT在整個磷脂質及三酸甘油酯的生合成過程扮演其相當程度的角色。根據其酵素活性與序列的相似性得知大約有9種亞型 (isoforms)，這些AGPAT都具有2到4個細胞嵌合區(transmembrane domains)和acyltransferases中高度保留的催化活性區(catalytic motif, NHX4D)以及受質結合區(substrate binding motif, EGTR)。Docosahexanoic acid (DHA, 22:6n-3)，為一長鏈n-3不飽和脂肪酸，在腦及視網膜的磷脂質sn-2位置上含量比起身體其他組織高出許多。相關的研究也指出DHA能維持神經細胞的正常功能，但是我們仍然不知道為何DHA選擇性結合到這些組織磷脂質的sn-2位置上。以酵素作用看來，AGPAT在sn-2脂肪酸的酯化扮演重要角色，九種AGPAT isoforms在不同組織表現量有很大的差別，因此認為各種isoform可能具有受質特異性，對特定的脂肪酸或是LPA進行sn-2的acylation。在腦部AGPAT mRNA表現量最高是AGPAT4，因此我們推測AGPAT4可能與DHA在大腦的累積有關。
利用RT-qPCR的方式測得隨著年齡增長(胚胎、七天大、一個月大及十個月大)，大鼠大腦中AGPAT4的mRNA表現量跟著降低，利用氣相層析分析法(Gas chromatography, GC) 發現其DHA的含量也相對跟著降低；另外，在DHA缺乏的大鼠腦部AGPAT4的表現，相較於控制組的表現量來的高。為了探討AGPAT4的功能，我們建立了含有大鼠AGPAT4 cDNA的表現質體並轉染到SH-SY5Y細胞，Western blot 及螢光免疫染色法可以看出AGPAT4的表現及其在細胞中之分佈。在轉染AGPAT4的細胞給予50μM的各種脂肪酸24小時，當給予DHA及α-linolenic acid (ALA, 18:3n-3)時，細胞中DHA及ALA相較於控制組分別高出3.0%及2.8%；但是當給予eicosapentaenoic acid (EPA)、 arachidonic acid (AA)、palmitoleic acid (PA, 16:1)和 miristic acid (MA, 14:0)細胞中AA、EPA、PA及MA含量則沒有明顯的改變。當分別給予10μM 及50μM DHA，轉染AGPAT4 cDNA 的細胞比只轉染載體的細胞多了1% 及 3%。用retinoic acid (RA) 及mitotic inhibitors分化SH-SY5Y成神經細胞， AGPAT4 mRNA表現量會隨著分化而增加，DHA的累積也有增加的趨勢，最高達1.76倍 (由2.7%提到至4.7%)。為了更進一步證明AGPAT4對於DHA在細胞膜累積的重要性，我們利用RNAi 干擾技術減少AGPAT4的表現，當給予50μM DHA時，累積在磷脂質的DHA比起對照組低了4-8%的含量。本實驗的結果顯示，AGPAT4在神經發育及DHA在神經細胞的累積扮演重要角色。 | zh_TW |
| dc.description.abstract | 1-acyl-sn-glycerol-3-phosphate acyltransferases (AGPATs), also known as lysophophatidic acid acyltransferase (LPAAT), are involved in the transfer of fatty acid, mostly unsaturated, to the sn-2 carbon during de novo synthesis of glycerolipids, such as phospholipids and triacylglycerol. At least nine isoforms of AGPATs have been identified based on activity and/or sequence similarity. All of them have two conserved motifs, NHX4D and EGTR. It is well known that the sn-2 fatty acid of phospholipids of the frontal cortex, hippocampus and retina of human and most animals are rich in docosahexainoic aicd (DHA, 22:6n-3). Accumulating evidences showed that DHA is important for the functions of neurons. Since AGPAT is responsible for the esterification of sn-2 position of lysophosphatidic acid(LPA), there may have a tissue specific AGPAT that responsible for the accumulation of DHA in brain. Among all the AGPATs, the AGPAT4 mRNA is mostly expressed in brain. We therefore speculate that AGPAT4 may involve in accumulation of DHA in the brain.
To investigate the relationship of AGPAT4 on DHA accumulation in neurons, the expression levels of AGPAT4 mRNA and DHA content in rat brains were determined. Results showed that both AGPAT4 mRNA and DHA content in the brain are decreased with increasing of age. Moreover, the level of AGPAT4 mRNA of the DHA deficient rats was higher than that of the control rats. In order to investigate the function of AGPAT4, an expression plasmid containing rat AGPAT4 cDNA was constructed and transfected into SH-SY5Y cells. After transfection, the cells were given with a variety of fatty acids (FAs) for 24h and the FA composition in the cell was analyzed by gas chromatography (GC). Compared with control cells, SH-SY5Y cells transfected with AGPAT4 incorporated higher DHA and α-linolenic acid (ALA, 18:3n-3) but not eicosapentaenoic acid (EPA), arachidonic acid (AA), palmitoleic acid (16:1), and miristic acid (14:0) compared with that in cells transfected with vector. After incubating with 0-50μM of DHA for 24 hours, the incorporation of DHA in AGPAT4 transfected SH-SY5Y cells increased in a dose-dependent manner, and the variation between SH-SY5Y with or without AGPAT4 overexpression was gradually different. Moreover, we differentiated SH-SY5Y to a neuronal-like state using a combination of retinoic acid and mitotic inhibitors. Both of the expression level of Agpat4 and DHA incorporation increase during cell differentiation. Knockdown the AGPAT4 expression by specific siRNAs resulted in decreased DHA content of phospholipids in SH-SY5Y cell. Taken together, this study showed that AGPAT4 may play a important function in DHA accumulation in neurons and in the SH-SY5Y differentiation. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-14T16:43:13Z (GMT). No. of bitstreams: 1 ntu-97-R95442003-1.pdf: 2310423 bytes, checksum: c17b0abbb3677f206c52d652ea5d5bf5 (MD5) Previous issue date: 2008 | en |
| dc.description.tableofcontents | 謝誌 iii
摘要 iv Abstract vi 縮寫對照表 viii 第一章 緒論 1 第一節 文獻回顧 2 第二節 研究動機與實驗目的 12 第二章 實驗材料與方法 14 I. 動物實驗 15 第一節 動物飼養、組織取樣及處理 16 一、 實驗動物與飼養條件 16 二、 動物犧牲 16 第二節 大鼠腦組織基因mRNA表現量測定(一) 16 一、 腦組織總RNA抽取 16 二、 第一股cDNA的合成 17 三、 反轉錄酶-聚合酶連鎖反應 (Reverse Transcriptase- Polumerase Chain Reaction, RT-PCR) 17 四、 DNA洋菜膠體電泳 18 第三節 大鼠腦組織基因mRNA表現量測定(二) 19 一、 引子設計 19 二、 定量聚合酶連鎖反應 (Quantitative-Polymerase Chain Reaction, Q-PCR) 19 第四節 脂肪酸組成檢測分析 20 第五節 統計分析 21 II. 細胞實驗 22 第一節 細胞培養 23 第二節 製備含有大鼠AGPAT4/myc-His表現質體的人類神經細胞株SH-SY5Y 23 第三節 分析在大量表現AGPAT4的SH-SY5Y細胞中蛋白的表現 28 第四節 建構不表現myc-His蛋白的pcDNA3.1-AGPAT4 表現質體 33 第五節 分析不同脂肪酸在細胞中的堆積情形 34 第六節 RNA干擾knockdown AGPAT4 35 第三章 實驗結果 38 第四章 討論 44 第五章 圖表 55 第六章 參考文獻 67 | |
| dc.language.iso | zh-TW | |
| dc.subject | SH-SY5Y | zh_TW |
| dc.subject | AGPAT4 | zh_TW |
| dc.subject | 分化 | zh_TW |
| dc.subject | 神經細胞 | zh_TW |
| dc.subject | SH-SY5Y | en |
| dc.subject | AGPAT4 | en |
| dc.subject | neuron | en |
| dc.subject | differentiation | en |
| dc.title | 大鼠腦組織及人類神經細胞株SH-SY5Y表現之1-acyl-sn-glycerol-3-phosphate acyltransferase的受質特性及年齡對其基因表現影響之探討 | zh_TW |
| dc.title | Studies on the substrate specificity and the effects of age on gene expression of 1-acyl-sn-glycerol-3-phosphate acyltransferase in rat brains and in SH-SY5Y cells | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 96-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 黃伯超(Po-Chao, Huang),吳文惠,蘇慧敏(Hui-Min, Su),盧志峰(Jyh-Feng, Lu) | |
| dc.subject.keyword | AGPAT4,SH-SY5Y,分化,神經細胞, | zh_TW |
| dc.subject.keyword | AGPAT4,SH-SY5Y,differentiation,neuron, | en |
| dc.relation.page | 76 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2008-08-01 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 生物化學暨分子生物學研究所 | zh_TW |
| 顯示於系所單位： | 生物化學暨分子生物學科研究所 | |
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